Medycyna Wet. 2006, 62 (1) 43
Praca oryginalna Original paper
According to the statistics from the United Nations Organization, approximately 40 to 45% of pig feeds worldwide are contaminated with mycotoxins (1). Two factors comparatively mild and wet climate of Lithuania and relatively not widely spread application of chemicals to growing plants, create favorable con-ditions for proliferation and spread of various fungi and their toxins into grain and feed supplies. Fusarium species of fungi and their toxins were prevailing in the feed grain of the 1998-1999 harvests of wheat and barley (Feed quality test laboratory Labtarna, Vilnius). The content of zearalenone in the feed grain was from 0.14 to 1.8 mg/kg, with the average being 0.6 mg/kg. The effect of this toxin on the animal depends on the concentration of the toxin present, duration of expo-sure, animal age and health status. Low concentrations of zearalenone in the feeds did not cause any signifi-cant health disturbances, however, negative effects on the reproductive performance of boars were observed. Of late years, wide application of mycotoxin de-toxication products has been started. The product Toxy--Nil Plus Dry (INVE NutriAd, Belgium) has an
adsor-bing effect on some types of mycotoxins (aflatoxin B1, ochratoxin A) and a biotransforming effect (zearale-none, all types of trichocetenes) on less harmful meta-bolites.
The aim of the present study was to determine the effects of mycotoxin zearalenone (the most common in our feeds) on the health status of boars and quality of their semen and also the effect of the detoxicating product Toxy-Nil Plus Dry (further in the text TNPD) on the quality of boar semen.
Material and methods
Thirteen Lithuanian White boars of 10 months of age and 150 to 155 kg weight were used in the experiment and allotted to three groups: control group (boars fed myco-toxin-free feeds; n = 3), experimental 1 (boars fed com-pound feed containing 0.57 mg/kg of zearalenone; the daily intake of this toxin amounted to 3.42 mg; n = 5) and experimental 2 (boars fed 0.57 mg/kg zearalenone con-taining feeds which, before feeding, were detoxicated with the product TNPD at a rate of 1 kg per 1000 kg of feed; n = 5). The boars in all groups were fed twice daily at
Effects of zearalenone and product Toxy-Nil
Plus Dry on physiological responses of boar semen
JONAS KUTRA, ARTÛRAS ÐIUKÐÈIUS, ALGIRDAS URBÐYS, VIDMANTAS PILECKAS, RASA NAINIENË, AUDRONË MANKEVIÈIENË
Institute of Animal Science of Lithuanian Veterinary Academy, R. ebenkos 12, LT-82317, Baisogala, Lithuania
Kutra J., Ðiukðèius A., Urbðys A., Pileckas V., Nainienë R., Mankevièienë A.
Effects of zearalenone and Toxy-Nil Plus Dry on physiological responses of boar semen Summary
The aim of the present study was to determine the effects of the mycotoxin zearalenone as well as the detoxicating product Toxy-Nil Plus Dry (TNPD) on the quality of boar semen. Thirteen Lithuanian White boars, 10 months of age and weighing 150 to 155 kg were used in the experiment and allotted to three groups. Every boar was offered 6 kg of compound feed containing 78% of barley meal and 22% of protein-vitamin--mineral premix (PVMP). Boars in the experimental groups were given feed containing zearalenone (0.57 mg/ /kg) for 32 days and detoxicated (1 kg TNPD per 1000 kg of feed) feeds. In a week following the administration of zearalenone-containing feeds to the boars, the volume of ejaculation had decreased by 40.8 % (P < 0.005) compared with the control group. The lowest initial spermatozoa motility in the semen collected during intoxi-cation was determined in the group of boars receiving zearalenone-containing feed (3.9 ± 1.79 points, P = 0.01). On replacement of the contaminated feed, the motility of spermatozoa recovered within a week and amounted to 7.0 ± 0.55 points. The negative effect of TNPD on spermatozoa was noted during the recovery period. The total quantity of pathologic spermatozoa in a corresponding experimental group increased to 33.2 ± 8.75% compared with 21.7 ± 8.27% (P < 0.05) in the pre-experimental period. The study indicated that the investigated level of zaeralenone in feed negatively affects the reproductive performance of boars. The unhealthy effect of zearalenone may be significantly reduced by treating contaminated feeds with TNPD.
Medycyna Wet. 2006, 62 (1) 44
7 a.m. and 4 p.m. The boars were offered 6 kg of com-pound feed containing 78% of barley meal and 22% of pro-tein-vitamin-mineral premix.
The whole experiment was divided into three periods: experimental, intoxication and recovery. During the pre--experimental period the boars were fed high quality feeds and trained to secrete semen by the manual method. Quali-tative and quantiQuali-tative parameters of semen have been eva-luated at the start, days 4 and 10. The length of the period was 10 days. At the period of intoxication, boars in the experimental groups were fed zearalenone-containing (experimental 1) and detoxicated (experimental 2) feeds. Semen from the boars in all groups was collected manually five times once a week starting on day 7. Physiological responses of semen were determined. The length of the period was 32 days.
The boars in all groups were fed only high quality feeds during the period of recovery. Physiological responses of semen were evaluated three times once a week conti-nuously beginning from the period of intoxication. The length of the period was 21 days.
The level of testosterone in the animal was determined by the blood analysis from v. cava cranialis. Ten milliliters of blood was taken at the end of the pre-experimental period, four times during the period of intoxication and two times during the period of recovery from the boars in all groups. The content of mycotoxins was analysed at the ac-credited Feed Quality Test Laboratory Labtarna (Vilnius). The amount of other fungeous toxins did not exceed the EU requirements, except for zearalenone.
The concentration of spermatozoa was determined with the binocular microscope Olympus BH using × 150 magni-fication. Spermatozoa were counted in Goryaevs chamber after dilution for 20 times. The concentration was estima-ted according to the formula:
K = S × P × 5 · 105,
where: K sperm count per mL semen, milliard spermato-zoa; S sperm count per 80 little square; P dilution level.
Motility of spermatozoa was determined in fresh semen and semen stored at +16°C for 72 h. The percentage of spermatozoa with progres-sive movement was determined and evaluated on a 10-point scale. The semen was diluted with the diluent containing (per 1,000 mL) 2.6 g of trilon B, 3.4 g of sodium citrate, 0.5 g of NaHCO3, 40.0 g of glucose, 1.8 g of (NH4)2SO4, 500,000 IU of penicillin and 0.5 g of strepto-mycin. The number of live and pathologic sper-matozoa per ejaculation was determined with the eosin dyes by melting 3 g of sodium citrate and 1.7 g of eosin per 100 mL of distilled water. For determination of the percentage of pathologic spermatozoa, a smear was prepared from fresh semen and analyzed with a micro-scope using × 150 magnification. The patholo-gy of proximal and distal drops, heads, middle part and tails of spermatozoa have been deter-mined. The condition of acrosomes was eva-luated by studying semen fixed in formalin so-lution with the phase-contrast microscope (5).
Centrifugation of blood lasted for 10 minutes at 400 × g to prepare the samples for testosterone level determination. Blood plasma (5 mL) was poured into test tubes and stored at 18°C. The level of testosterone in blood plasma was determined by the radioimmune method at the Radio-immune Laboratory of the Institute of Endocrinology in Kaunas using the Testo-RIA-CT kit (BioSource, Belgium) with a minimal detectable concentration of 0.044 ± 0.009 ng/mL. The coefficients of variation for intra- and inter-assay variability were 4 to 4.7% and 8.1 to 8.3%, respecti-vely.
Data sets of both experimental 1 and experimental 2 groups were investigated independently and compared with the control group in each period correspondingly, be-cause the treatments in experimental groups were carried out towards the exacerbation and betterment, respecti-vely. Therefore, the differences in the control group were compared using the Microsoft Excel 2000 t-test data ana-lysis tool. The variances of compared means were assumed homogeneous when their inter-ratio was less than 3 in any combination. A probability value of P < 0.05 was used as an indicator of statistical significance among comparisons.
Results and discussion
In a week after the start of feeding zearalenone-con-taining feeds to boars, the volume of ejaculation has decreased by 40.8% (P < 0.005) compared with the control group and amounted to 141.0 ± 12.59 mL. At week 2 of intoxication, the volume was still reduced, however, later it started increasing (fig. 1). On com-pletion of the experimental feeding, the volume of ejaculation was recovered in a weeks time and during the last week of recovery the volume of ejaculation was even higher than that of the control group. It has been reported that the changes of the sperm volume per ejaculation are dependent on the testosterone con-centration in blood (2, 4). In our study, the concentra-tion of testosterone in the blood of experimental boars
130 150 170 190 210 230 250 1 10 19 28 37 46 55 64 Trial period, d Volume, mL Control With zearalenone With TNPD
Fig. 1. Time course of changes in the volume of ejaculate of boars
Explanations: the vertical lines indicate the boundaries of the pre-experimental, chronic intoxication and recovery periods. Boars in groups (n = 5) were fed with 0.57 mg/kg zaeralenone-containing (experimental 1; mentioned as With zaeralenone) and detoxificated with product TNPD (experimental 2 as With TNPD) feed from 10th till 42nd day of trial.
Medycyna Wet. 2006, 62 (1) 45 was increased (fig. 2) and might have influenced the
sperm volume per ejaculation.
At week 2 of intoxication, lower sperm volume per ejaculation was accompanied by lower total count of
spermatozoa per ejaculation (24.1 ± 0.73 vs. 51.4 ± 6.70 milliard spermatozoa in control group, P < 0.001; fig. 3). This is in agreement with the findings of (7).
During the intoxication period, the spermatozoa count per ejaculation in group experimen-tal 1 was lower, yet there was no significant difference between the control and experi-mental 2 groups (fig. 3). On replacement of the detoxicated feed with the quality one, the total count per ejaculation amounted to 56 to 69 milliard spermatozoa during the first week of recovery and remained con-stant till the end of the experiment.
The lowest initial spermatozoa motility in the semen was determined at the end of the intoxication period in the group of boars fed zearalenone-containing feed (3.9 ± 1.79 vs. 7.9 ± 0.19 points in control group, P < 0.05). After replacement of the contaminated feed, the motility of sperma-tozoa has recovered already at week 1 and amounted to 7.0 ± 0.55 points (fig. 4). Lower motility of spermatozoa could be associa-ted with either sperm membrane damage or metabolism disturbances in the cell. The sperm motility of the boars fed TNPD treated feed did not differ significantly from that of the control group.
During the first week of intoxication, when semen was diluted and stored at +16°C for 72 hours, sperm motility in all groups was on level of 4 points, however, at week 5 of zearalenone feeding, this indi-cator lowered to 1.8 ± 0.57 vs. 5.0 ± 0.50 points (P < 0.001) in the control group. The usage of TNPD indicated that sperm moti-lity at week 5 of intoxication was 3.9 ± 1.23 points after 72 h of storage (data of this pa-ragraph not shown).
The survival time of spermatozoa in the diluted semen of all boar groups was high during the whole intoxication period. The spermatozoa remained viable up to 295.3 ± 50.53 h in the group of boars fed zearale-none-containing feed and up to 332.4 ± 3.19 h in the group of boars fed TNPD de-toxicated feed (data not shown). This indi-cator in the control group was similar. Du-ring the period of recovery, there was no difference for the survival time of sperma-tozoa between both experimental and con-trol groups.
Pathologic spermatozoa, especially those with the coiled tails have been found in the semen of boars in all three groups (fig. 5). Higher pathology of tails in the experimen-tal groups could be determined due to func-tional disorders of the testicular epididymis
0 1 2 3 4 5 6 1 10 19 28 37 46 55 64 Trial period, d Concentration, ng/cm 3 Control With zearalenone With TNPD
Fig. 2. The quantitive changes of testosterone concentration in the course of the trial
Explanation: arrows point to the start and the end of feeding of experimental feeds 20 30 40 50 60 70 80 1 10 19 28 37 46 55 64 Trial period, d Spermatozoa count, milliard Control With zearalenone With TNPD
Fig. 3. Quantitive changes of the spermatozoa count per ejaculation du-ring the trial
Explanations: see the explanations to fig. 1.
3 4 5 6 7 8 9 1 10 19 28 37 46 55 64 Trial period, d Motility score, points Control With zearalenone With TNPD
Fig. 4. The quantitive changes of initial spermatozoa motility in the course of the trial
Medycyna Wet. 2006, 62 (1) 46
(7). The number of spermatozoa with acro-somal defects, proximal and distal drops, pathology of heads was higher (differences not significant) in both experimental groups compared with the control what has been also confirmed by (6).
The negative effect of TNPD on sperma-tozoa has been revealed during the recove-ry period. The total quantity of pathologic spermatozoa in experimental 2 group has increased to 33.2 ± 8.75% in comparison with 21.7 ± 8.27% (P < 0.05) in pre-experi-mental period. Spermatozoa tail damages were prevailing.
During the period of intoxication, the concentration of testosterone in the blood of boars fed zearalenone-containing feed was increased (fig. 2) and that could be due to stimulation of the sexual functions of boars during semen collection. Higher
con-centration of testosterone could be also found due to zearalenone effect on the neuroendocrine system through higher LH content in blood, which stimulates testosterone release from Leidig cells. Higher than normal concentration of testosterone in blood affects Sertoli cells found in the testicles, which, consequent-ly, produce more pathologic spermatozoa and, thus reduces the physiological indicators of semen (3).
The study indicated that even a month-length feeding of pigs with up to 0.57 mg/kg zaeralenone containing feeds might not reveal the clinical symptoms of body intoxication. However, this amount of zearalenone negatively affects the reproductive performance of boars, i.e. the physiological responses of semen be-come worse. The unhealthy effect of zearalenone may be significantly reduced by treatment of contaminated
Fig. 5. The differences between the content of pathologic spermatozoa in the sperm of boars
Explanations: large bar indicates the overall pathology of all kinds of sperm. All differences are not significant except that between the values of all pathology in pre-experimental and recovery period in a group fed with TNPD (P < 0.05).
0 5 10 15 20 25 30 35 40 With
zearalenoneWith TNPD Control zearalenoneWith With TNPD Control zearalenoneWith With TNPD Control
Preexperimental- Experimental Recovery
Trial period
All Tails Drops Heads Acrosomes Other
Pathologic
spermatozoa,
%
feeds with product TNPD. However, it is advisable to use only high-quality feeds for boar feeding.
References
1.Alltech: Mycotoxin Monthly. Inf. Bull., (ed.): Alltech, Inc., Nicholasville, KY. 2000, 3, 3.
2.Espey L. L.: Mechanisms of immunoregulation in normal human pregnancy. Am. J. Reprod. Immunol. Microbiol. 1988, 16, 81.
3.Holdstock G., Chactenay B. F., Kriwitt E. L.: Effects of testosterone, oestra-diol and progesterone on immune regulation. Immunology. 1988, 47, 449-457. 4.Howorka F., Slechta J.: Ejaculate volume and quality in boars aged 5, 6 and
7 months. Anim. Breed. Abstr. 1984, abstr. no. 2997.
5.Pursel V. G., Johnson L. A., Rampacek G. B.: Acrosome morphology of boar spermatozoa incubated before cold shock. J. Anim. Sci. 1972, 34, 278-283. 6.Stolla R., Bauer J., Gedek B.: Spermabeschaffenheit beim Eber nach
Verfut-terung des Mykotoxins Zearalenon. Zuchthygiene. 1987, 22, 165-172. 7.Young L. G., King G. J.: Low concentrations of zearalenone in diets of boars
for a prolonged period of time. J. Anim. Sci. 1986, 63, 1197-1200. Authors address: PhD Jonas Kutra, R. ebenkos 12, LT-82317 Baisogala, Radvilikio r., Lithuania; e-mail: reprodukcija@lgi.lt
